SIMULTANEOUS VOLTAMMETRIC COMPARISONS OF REDUCTION POTENTIALS, REACTIVITIES, AND STABILITIES OF THE HIGH-POTENTIAL CATALYTIC STATES OF WILD-TYPE AND DISTAL-POCKET MUTANT (W51F) YEAST CYTOCHROME-C PEROXIDASE

Protein film voltammetry has been used to measure changes in the catal ytic redox energetics of cytochrome c peroxidase produced by a single mutation in the distal pocket. Wild-type (WT) cytochrome c peroxidase adsorbs at a pyrolytic graphite edge electrode from ice-cold dilute su ccinate buffer, pH 5.4, to give an electroactive film showing a revers ible and narrow (two-electron) signal, reduction potential 754 mV, whi ch converts completely to a catalytic wave at a similar potential when low levels of hydrogen peroxide are added. Under the same conditions, the W51F mutant yields a weaker signal at 883 mV which also transform s to a catalytic wave at similar potential, but with amplitude compara ble to that of WT. Zn either case the catalytic rates are very high. T he reversible signals observed for each variant therefore correspond t o the catalytic redox couple, analogous if not identical to Fe-IV=O,R/Fe-III, with replacement of tryptophan-51 by phenylalanine causing a substantial increase in reduction potential (destabilization of Fe-IV= O,R+). The W51F variant appears less stable, even in the resting state , but this does not seriously undermine the results. When the two vari ants are studied in competition, the non-turnover voltammetry is domin ated by the greater electroactive coverage of the WT enzyme, whereas p eroxide reduction is controlled at all but the highest rotation rates by the more active W51F. The experiment provides a direct comparison o f the real (thermodynamic) catalytic efficiencies of redox enzymes, in this case clearly identifying W51F as intrinsically the more active a nd efficient variant (higher reduction rates at lower driving force).